The long term goal of this research is to understand the molecular mechanism of the complex developmental process by which Rhizobium meliloti forms symbiotic nitrogen-fixing nodules on the roots of its host alfalfa. Genetic and molecular biological strategies commonly used with Escherichia coli, lately made possible in this system, will be combined with biochemical, immunological and cell physiological techniques in a multidisciplinary approach based on the complementary expertise of two established laboratories. As specific aims, genetic tools will continue to be developed, including transposon Tn5 derivatives for making operon fusions in vivo, Hfr-like strains for conjugal mapping, "maxicells" for visualizing proteins, and host-range and conditional phage mutants for improved transduction. Genetic analysis of exopolysaccharide (EPS) required for effective nodulation will also continue, including cloning and characterization of genes, isolation of new mutants and identification of additional loci. Both extracellular products and lipid-bound biosynthetic intermediates of EPS-deficient mutants will be characterized biochemically. Morphological studies will further characterize the atypical "empty" nodules of EPS mutants, the interaction with plant roots of mutants having altered EPS, and, by immunofluoresence with monoclonal antibodies, the distribution of EPS in nodule tissue. The ability of isolated EPS to restore effective fixation to mutants will be analyzed further. Regulation of symbiotic genes by plant products will be approached by fusion of nodulation operons to easily assayed reporter genes. This system will serve as a model for the developmental interaction of prokaryotic and eukaryotic cells, of which there are many examples in the area of human health. More specifically, this work will help elucidate the mechanism of biological nitrogen fixation in legumes, with immediate relevance to the health-related issues of plant productivity, agronomic energy conservation and provision of nitrogen in the diet.